This invention relates to a new class of chemical compounds and their use in medicine. In particular the invention provides novel macromolecules, methods for their preparation, pharmaceutical formulations thereof and their use as anti-influenza agents. The invention also provides a novel diagnostic method which can be used for detection of all types of influenza A and B virus.
Influenza A and B viruses are major causes of acute respiratory disease, resulting in an estimated 30-50 million infections annually in the United States alone. Influenza A has been responsible for major epidemics, such as the xe2x80x9cSpanish Fluxe2x80x9d of 1919 which killed millions of people. Influenza remains a difficult disease to control, resulting in significant morbidity, and mortality largely due to secondary infection in eldery or debilitated patients. Vaccines are continually being rendered obsolete by antigenic shift or drift, and consequently immunization is only about 70% effective in preventing infection. The only drugs approved by regulatory authorities for treatment of influenza are amantidine and rimantidine, which are ineffective against influenza B, and are known to have serious side-effects.
Many viral and bacterial infections may present with symptoms similar to those of influenza. The rapid identification of respiratory viruses would enable physicians to use the most appropriate therapy early in the illness. For example, an early and accurate diagnosis would allow decisions regarding the use of antibacterial therapy and hospitalisation of children and the elderly.
Laboratory tests for the identification of viruses in clinical material are widely used, and a variety of different detection methodology is available. The textbook, xe2x80x9cLaboratory Diagnosis of Viral Infectionsxe2x80x9d, Marcel Dekker 1992, Ed E. H. Lennette generally discusses methods which are used for a wide range of viruses, including influenza virus.
A number of tests are available for the diagnosis of influenza A and B. The traditional method of identifying influenza viruses has been the use of cell culture, which is highly sensitive and specific. Unfortunately, the time required for culture, isolation and identification of influenza virus can range between 2 and 10 days, thus making it virtually useless in guiding the physician to an appropriate therapy. Since influenza virus infection is normally self-limited, diagnosis must be rapid if therapy is to be effective.
In addition to the cell culture methods for detecting influenza, there have recently become available a few rapid direct tests, which are specific for influenza A. Thus, a monoclonal immunofluorescence assay (IFA) has been reported (Spada, B. et al, J. Virol. Method, 1991 33 305) and at least one rapid enzyme immunoassay (EIA) is available (Ryan-Poirier, K. A. et al, J. Clin. Microbiol., 1992 30 1072). A number of comparisons of these rapid detection methods for influenza A have been reported; see for example Leonardi, G. P. et al, J. Clin. Microbiol., 1994 32 70, who recommended that direct specimen testing be used together with culture isolation, so as to permit both identification of the virus in time to institute therapy and infection control measures, and to monitor the antigenic constitution of influenza strains prevalent in the community.
The IFA method is reported to be labor-intensive, and requires considerable technical expertise, with the results often being difficult to interpret. On the other hand, the EIA (Directigen FLU-A; Becton Dickinson Microbiology Systems) method gave a high level of false-positive results, and it has been recommended that this assay should be used in laboratories only as an addition or substitute for direct immunofluorescence tests (Waner, J. L. et al, J. Clin. Microbiol., 1991 29 479).
As well as the problems mentioned above with the currently available rapid assays for influenza, there are other fundamental deficiencies in some of these methods. Firstly, none of the available assays can detect influenza B, which means that even a negative test result would leave the physician uncertain about the type of therapy that should be used. Secondly, if a rapid immunoassay method depends on the use of antibodies to one of the influenza A proteins, there may be a serious problem in detecting new strains of the virus which have undergone a drift or shift in the structure of the antigenic proteins. Influenza A is notorious for its propensity to undergo such changes.
Another type of rapid assay for influenza viruses has been described in a series of patent specifications (see for example Liav, A. et al, PCT Patent Application No. 92/12256). The method involves the use of a chromogenic substrate for the influenza neuraminidase enzyme. In other words the assay depends on visualising a dye, which is formed when the influenza neuraminidase cleaves a special sialic acid-dye conjugate molecule. This technique appears to offer limited specificity, because it could not readily distinguish between the presence of viral neuraminidase and other forms of the enzyme, particularly bacterial neuraminidase. It may also have low sensitivity because of the relatively slow activity of viral neuraminidase.
Influenza A and B have two major surface glycoproteins, hemagglutinin (HA) and the enzyme neuraminidase (NA), which are both essential for infectivity. It is believed that HA is necessary for the virus to attach to cells whereas NA is needed for release of the virus from cell surfaces. There are typically about 600 trimeric HA and about 50 copies of the NA tetramer units on the surface of each virus particle. Both HA and NA therefore are attractive potential targets in the search for anti-influenza drugs, but to date no anti-influenza drugs that work at either of these sites are available for clinical use.
Influenza virus hemagglutinin binds to the sialic acid containing glycoproteins and glycolipids on cell-surface receptors, thereby initiating the process of attachment of the virus to a cell and subsequent infection. The strength of the binding of a virus particle to the cell membrane appears to depend on the interaction of multiple copies of the influenza HA with multiple sialic acid groups on the cell surface.
Using this concept of a polyvalent interaction, several workers have reported the synthesis of macromolecules containing two or more sialic acid derivatives which act as hemagglutinin inhibitors. Although some strong HA inhibitors have been discovered, none of these polyvalent macromolecules has been shown to prevent influenza infection in vivo. Recent papers by Whitesides and co-workers (J. Amer. Chem. Soc., 1996 118 3789-3800; J. Medicinal Chem., 1995 38 4179-4190) have summarised the various efforts which have used this approach to the design of inhibitors of influenza hemagglutinin.
There are several known inhibitors of NA, most of which are close analogues of neuraminic acid, the enzyme""s natural substrate, such as 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) (Meindl et al, Virology, 1974 58 457-63). International Patent Application No. WO 91/16320 describes analogs of DANA which are very active, both in vitro and in vivo, against influenza A and B neuraminidase. One of these compounds (Compound I, designated GG167 or 4-guanidino-Neu5Ac2en) is in clinical trial, and shows promise for the treatment of influenza (Hayden, F. G. et al, J. Amer. Med. Assoc., 1996 275 295). 
More recently, aromatic compounds with neuraminidase-inhibitory activity have been described in U.S. Pat. No. 5,453,533 by Luo et al and U.S. Pat. No. 5,512,596 by Gilead Sciences, Inc., and analogues of compound (I), in particular compounds in which the side-chain at carbon 6 is ether-linked, have been described in International Patent Application No. WO 96/26933 by Gilead Sciences, Inc. and in C. Kim et al, J. Amer. Chem. Soc., 1997 119 681.
Several research groups have attempted to find simpler or more potent analogues of compound (I), but reports to date (e.g. Bamford M. J., J. Chem. Soc. Perkin Trans. I, 1995, 1181) indicate that any changes to the structure of compound (I), particularly at the glycerol side chain, are likely to reduce the neuraminidase-binding properties. In addition, in contrast to the situation with HA, there do not appear to be any known macromolecular or polymeric inhibitors of neuraminidase. Sialic acid-containing polymers have been described in U.S. Pat. No. 5,192,661 by Roy et al and in U.S. Pat. No. 5,571,836 by Bovin et al, but these compounds were synthetic polysialosides designed for use as antigens or as binders of haemagglutinin.
In a first aspect, the invention provides macromolecular compounds which have attached to them one or more molecules which bind to the active site of influenza virus neuraminidase; these molecules are referred to herein as xe2x80x9cneuraminidase bindersxe2x80x9d. Preferably the neuraminidase binder is attached to the molecule via a spacer or linker group so that the neuraminidase binder is not sterically hindered by the backbone of the macromolecule. The neuraminidase binder may be any agent which binds to the active site of influenza virus neuraminidase, provided that it is not cleaved by the enzyme. The binding need not be irreversible, but the binding group should have a high binding affinity, preferably an IC50 of 10xe2x88x926 M or less.
The invention particularly relates to a new class of chemical compounds and their use as therapeutic and diagnostic agents for the treatment and detection of influenza A and B. More specifically the invention concerns macromolecules which have attached to them neuraminic acid (sialic acid) derivatives which bind to neuraminidase of influenza A or B, and which optionally also have a functionality which allows the compounds to be bound to a surface, or which can be used as a detectable label.
Surprisingly, we have found that when compound (I) is functionalised through the 7-position of the sialic acid structure, it can be attached to large synthetic or natural polymers to give complexes which inhibit influenza A and B neuraminidase, and which can prevent or inhibit influenza infection. Rather than destroying the influenza neuraminidase-binding properties of compound (I), we find that when multiple numbers of this and similar compounds are linked through their 7-position by a suitable spacer to a variety of macromolecules the average binding per sialic acid group is not substantially reduced. Thus through the binding to neuraminidase the macromolecules are tightly bound to the virus, and possibly because of the size and steric effects of the complexes, the infectivity of the influenza virions is reduced. Such macromolecular compounds can also be used to enable the detection of influenza A and B virus through their ability both to bind the influenza virus selectively and at the same time to be bound to a surface or to a detectable linking group.
The biological activity of the macromolecular compounds of the invention and the diagnostic method of the invention are both based on the use of ligands on the macromolecules that are able to bind specifically to the active site of influenza virus neuraminidase, or functionalised derivatives of such compounds, as binding and/or detecting agents to identify influenza virus in clinical specimens. The term xe2x80x9cneuraminidase bindersxe2x80x9d is used hereinafter to refer to these compounds and their functionalized derivatives. The method and compounds of the invention can function either in the presence or the absence of compounds binding non-specifically to influenza virus neuraminidase.
In a preferred embodiment, the present invention provides a compound of formula (II):
(Xxe2x80x94Y)nxe2x80x94Mxe2x80x94(Z)mxe2x80x83xe2x80x83(II)
wherein X is a neuraminidase-binding 2,3-dehydro-sialic acid derivative (2) which is linked at the 7-position via a spacer group Y to a macromolecule M, and Z is an optional extra substituent on the macromolecule.
The neuraminidase-binding moiety X is a sialic acid derivative of formula (2) 
in which the spacer Y connects to the W group, and
wherein R represents an azido group, an unsubstituted or substituted guanidino group, or an unsubstituted or substituted amino group;
R2 represents COCH3, COCF3, SO2CH3 or SO2CF3;
W represents O(Cxe2x95x90O)NH, O(Cxe2x95x90S)NH, NH(Cxe2x95x90O)NH or NH(Cxe2x95x90S)NH and is attached through the NH to group Y;
m is an integer between 0 and 1000; and
n is an integer between 1 and 1,000.
The spacer group Y is an optionally substituted chain of up to 1000 atoms chosen from carbon, nitrogen, oxygen and sulphur.
The macromolecule M is a synthetic or natural polymer, protein, antibody or enzyme of molecular weight from 104 up to 107.
The Y group is generally linked covalently to the macromolecule M, but may also be bound through non-covalent attachment, for example when M is avidin and Y has a terminal biotin group.
The second and optional substituent Z can be a group that binds hemagglutinin, such as a 2-linked sialic acid derivative, or a group that can act as a detectable label, such as a biotin or fluorescent molecule, or it can be an antibody-binding hapten. The optional substituent z can also be an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) which can be used to enable detection of influenza. Alternatively, the group Z can be a group with a terminal functionality that is suitable for binding the macromolecule to a surface, such as NH2, SH, CO2H, CHO, or CHxe2x95x90CH2.
In another preferred embodiment, the invention provides neuraminidase binders of formula (IIA):
(Xxe2x80x2xe2x80x94Y)nxe2x80x94Mxe2x80x94(Z)mxe2x80x83xe2x80x83(IIA)
wherein Xxe2x80x2 is a neuraminidase-binding cyclohexenyl derivative of formula (2a), 
which is linked through the ether side-arm,
R, R2,Y, n and m are as defined above for formula (2), and
R1 and Wxe2x80x2 are lipophilic C1-C12 alkyl or alkylene groups which are optionally substituted by one or more halogen atoms or alkoxy, haloalkoxy or optionally substituted aryl groups.
Suitable spacer groups Y include, but are not limited to, aminoalkyl groups, (poly)amino acids, linear peptides, oligosaccharides and polysaccharides, polyethylene glycol units, and amino-dialkylureas, any of which may be used alone or in combination. Typically the spacer group Y has a terminal amino group, which is used to form an amide or Schiffs base linkage on to the macromolecule M.
Suitable substituents of the guanidino or amino groups R include methyl, ethyl, allyl, amino, cyano or nitro.
Suitable macromolecules M include proteins, enzymes, antibodies, water-soluble synthetic polymers such as polyacrylic acids and polyacrylamides, polysaccharides and polyaminoacids. Macromolecules which are particularly suitable for use in diagnostic applications include bovine serum albumin (BSA), horseradish peroxidase (HRP), avidin and related proteins such as streptavidin or neutravidin, and immunoglobulins.
Macromolecules which are particularly suitable for use in compounds of the invention to be used in the treatment of influenza include polysaccharides, synthetic polymers such as polyacrylamides, polyethylene glycols, polyureas, polyacids, polyesters, polyamides and various co-polymers such as N-(2-hydroxy-propyl)methacrylamide (HMPA), which are known to be safe for administration to humans. The person skilled in the art will be aware of other pharmaceutically-acceptable polymers.
One preferred group of compounds of the invention comprises compounds (II), in which X is a GG167 derivative of formula (2) wherein:
R is guanidine, R2 is acetyl, W is the group O(xe2x95x90CO)NH and the spacer Y is a chain made up of between 6 and 60 carbon, nitrogen and oxygen atoms.
The macromolecules of formula (II) are inhibitors of influenza A and B neuraminidase and possess anti-influenza activity. Thus in a second aspect of the invention there is provided a pharmaceutical composition for treatment of influenza A or influenza B comprising a compound of the invention, preferably a compound of formula (II) or formula (IIA), or a pharmaceutically-acceptable derivative thereof, together with a pharmaceutically-acceptable carrier.
In a third aspect there is provided a method of treatment of influenza infection in a mammal, including man, comprising the step of administering an effective amount of a compound of the invention, preferably a compound of formula (II) or formulae (IIA) or a pharmaceutically acceptable derivative thereof, to a mammal in need of such treatment.
There is also provided in a fourth aspect the use of a compound of the invention, preferably a compound of formula (II) or formula (IIA) for the manufacture of a medicament for the treatment of an influenza viral infection.
The compounds of the invention may also be used in combination with other therapeutic agents, for example other anti-infective agents, particularly other anti-viral agents. The invention thus provides in a further aspect a pharmaceutical composition comprising a compound of the invention, preferably a compound of formula (II) or formula (IIA) or a pharmaceutically acceptable salt or derivative thereof together with one or more further therapeutically active agent, in particular an anti-viral agent, together with a pharmaceutically-acceptable carrier.
The invention also provides in another aspect a method of detection of influenza virus, comprising the step of exposing a sample suspected to include said virus to a compound of the invention which is able to bind specifically to the active site of influenza virus neuraminidase.
The method of the invention is applicable to all types of influenza A and influenza B.
For the detection of influenza the compounds of the invention (II) may be attached to a surface, either by covalent bonding or by non-specific binding. The spacer group Y should be sufficiently long that the neuraminidase binding units X are exposed on the surface of the imacromolecule M and accessible to a virus particle.
For the detection of influenza the method of the invention may use selective capture with a compound of formula (II) and thereby concentration of the virus, followed by detection of the virus using any convenient conventional method; the detection method need not have inherent selectivity. For example, the binder (II) may be attached to a support material, such as a membrane or polymer, such that virus particles will be selectively captured and concentrated when a sample is passed over or through the support. Therefore in one preferred embodiment of the invention, the group Z terminates in a functionality able to bind to a surface. Many suitable functionalities are known in the art.
Alternatively, a selective detection approach may be used; the virus particles in a sample may for example be non-specifically captured and then exposed to a macromolecular neuraminidase binder (II) which includes a detectable label Z, under conditions such that the binder attaches selectively to the influenza neuraminidase on the surface of the viral particle. The detectable label is then detected using any convenient method. For some detection systems, it is convenient to focus the sample into a confined area, for example a spot or a line on a surface. This may be achieved by a variety of methods; for example, the sample may be suspended or non-selectively captured on to a filter or other support material, and then exposed to the labelled binder as above.
In another alternative aspect the invention may use a combination of selective capture and selective detection to provide a simple and sensitive two-stage method of detecting influenza virus. This makes use of the fact that influenza virus particles typically have about one hundred neuraminidase molecules spread over their spherical surface (White, D. O., Curr. Top. Microbiol. Immunol., 1974 63 1-48), and can therefore attach to more than one binder at the same time.
Thus, a neuraminidase binder compound (II) may be attached to a support, for example as a narrow band across a length of porous membrane. The test sample is then applied at the other end of the membrane and allowed to flow across the band of bound compound. Any influenza virus particles in the test sample will be trapped by the membrane-bound compound (II) and thus retained in the narrow band. In the second stage of the test, a detectable label attached to another neuraminidase binder (II) is allowed to flow through the membrane across the band of bound influenza virus particles. The presence of influenza virus is then shown by an observable change in the membrane at the site of the bound compound. It is contemplated that the method and compounds of the invention are suitable for use with the Biostar Optical Immunoassay (OAI) platform, which is described inter alia in U.S. Pat. No. 5,418,135 by Miller et al.
A very large number of suitable detection systems is known in the art, for example biotin-streptavidin, enzymic systems such as horseradish peroxidase or alkaline phosphatase, fluorescence systems, chemiluminescence systems, colloidal gold, radioactive labels and agglutination systems. It is contemplated that colloidal gold coated with a compound of the invention (II) will be a particularly convenient detectable label. Similarly, compounds of the invention wherein the macromolecule M is horseradish peroxidase are expected to be ideal for the ready detection of influenza. The skilled person will readily be able to select a suitable detection system and to optimise conditions for detection, using normal trial and error experimentation.
The compounds of the invention of formula (II) and their pharmaceutically acceptable salts and derivatives may be prepared by various methods which include those described below. The methods of preparation outlined below form another aspect of the invention.